Screening for Abiotic Stress Response in Rice.

Rice (Oryza sativa L.) is the staple food for over half of the world population. However, most rice varieties are severely injured by abiotic stresses, with strong social and economic impacts. Understanding rice responses to stress may guide breeding for more tolerant varieties. However, the lack of consistency in the design of the stress experiments described in the literature limits comparative studies and output assessments. The use of identical setups is the only way to generate comparable data. This chapter comprises three sections, describing the experimental conditions established at the Genomics of Plant Stress (GPlantS) unit of ITQB NOVA to assess the response of rice plants to different abiotic stresses-high salinity, cold, drought, simulated drought, and submergence-and their recovery capacity when intended. All sections include a detailed description of the materials and methodology and useful notes gathered from our team experience. We use seedlings since rice plants at this stage show high sensitivity to abiotic stresses. For the salt, cold, and simulated drought (PEG, polyethylene glycol) stress assays, we grow rice seedlings in a hydroponic system, while for the drought assay, plants are grown in soil and subjected to water withholding. For submergence, we use water-filled Magenta boxes. All setups enable visual score determination and are suitable for sample collection during stress imposition and also recovery. The proposed methodologies are affordable and straightforward to implement in most labs, allowing the discrimination of several rice genotypes at the molecular and phenotypic levels.

Visualization of a curated Oryza sativa L. CDPKs Protein-Protein Interaction Network (CDPK-OsPPIN).

Calcium-Dependent Protein Kinases (CDPKs) translate calcium ion (Ca2+) signals into direct phosphorylation of proteins involved in stress response and plant growth. To get a clear picture of CDPKs functions, we must identify and explore the CDPKs targets and their respective roles in plant physiology. Here, we present a manually curated Oryza sativa L. CDPK Protein-Protein Interaction Network (CDPK-OsPPIN). The CDPK-OsPPIN provides an interactive graphical tool to assist hypothesis generation by researchers investigating CDPK roles and functional diversity.

Carbon/nitrogen metabolism and stress response networks - calcium-dependent protein kinases as the missing link?

Calcium-dependent protein kinases (CDPKs) play essential roles in plant development and stress responses. CDPKs have a conserved kinase domain, followed by an auto-inhibitory junction connected to the calmodulin-like domain that binds Ca2+. These structural features allow CDPKs to decode the dynamic changes in cytoplasmic Ca2+ concentrations triggered by hormones and by biotic and abiotic stresses. In response to these signals, CDPKs phosphorylate downstream protein targets to regulate growth and stress responses according to the environmental and developmental circumstances. The latest advances in our understanding of the metabolic, transcriptional, and protein-protein interaction networks involving CDPKs suggest that they have a direct influence on plant carbon/nitrogen (C/N) balance. In this review, we discuss how CDPKs could be key signaling nodes connecting stress responses with metabolic homeostasis, and acting together with the sugar and nutrient signaling hubs SnRK1, HXK1, and TOR to improve plant fitness.

Turning the Knobs: The Impact of Post-translational Modifications on Carbon Metabolism.

Plants rely on the carbon fixed by photosynthesis into sugars to grow and reproduce. However, plants often face non-ideal conditions caused by biotic and abiotic stresses. These constraints impose challenges to managing sugars, the most valuable plant asset. Hence, the precise management of sugars is crucial to avoid starvation under adverse conditions and sustain growth. This review explores the role of post-translational modifications (PTMs) in the modulation of carbon metabolism. PTMs consist of chemical modifications of proteins that change protein properties, including protein-protein interaction preferences, enzymatic activity, stability, and subcellular localization. We provide a holistic view of how PTMs tune resource distribution among different physiological processes to optimize plant fitness.

The interplay between Mn and Fe in Deinococcus radiodurans triggers cellular protection during paraquat-induced oxidative stress.

The bacterium Deinococcus radiodurans is highly resistant to several stress conditions, such as radiation. According to several reports, manganese plays a crucial role in stress protection, and a high Mn/Fe ratio is essential in this process. However, mobilization of manganese and iron, and the role of DNA-binding-proteins-under-starved-conditions during oxidative-stress remained open questions. We used synchrotron-based X-ray fluorescence imaging at nano-resolution to follow element-relocalization upon stress, and its dependency on the presence of Dps proteins, using dps knockout mutants. We show that manganese, calcium, and phosphorus are mobilized from rich-element regions that resemble electron-dense granules towards the cytosol and the cellular membrane, in a Dps-dependent way. Moreover, iron delocalizes from the septum region to the cytoplasm affecting cell division, specifically in the septum formation. These mechanisms are orchestrated by Dps1 and Dps2, which play a crucial role in metal homeostasis, and are associated with the D. radiodurans tolerance against reactive oxygen species.

Posttranslational Modification of the NADP-Malic Enzyme Involved in C4 Photosynthesis Modulates the Enzymatic Activity during the Day.

Evolution of the C4 photosynthetic pathway involved in some cases recruitment of housekeeping proteins through gene duplication and their further neofunctionalization. NADP-malic enzyme (ME), the most widespread C4 decarboxylase, has increased its catalytic efficiency and acquired regulatory properties that allowed it to participate in the C4 pathway. Here, we show that regulation of maize (Zea mays) C4-NADP-ME activity is much more elaborate than previously thought. Using mass spectrometry, we identified phosphorylation of the Ser419 residue of C4-NADP-ME in protein extracts of maize leaves. The phosphorylation event increases in the light, with a peak at Zeitgeber time 2. Phosphorylation of ZmC4-NADP-ME drastically decreases its activity as shown by the low residual activity of the recombinant phosphomimetic mutant. Analysis of the crystal structure of C4-NADP-ME indicated that Ser419 is involved in the binding of NADP at the active site. Molecular dynamics simulations and effective binding energy computations indicate a less favorable binding of the cofactor NADP in the phosphomimetic and the phosphorylated variants. We propose that phosphorylation of ZmC4-NADP-ME at Ser419 during the first hours in the light is a cellular mechanism that fine tunes the enzymatic activity to coordinate the carbon concentration mechanism with the CO2 fixation rate, probably to avoid CO2 leakiness from bundle sheath cells.

Exploring the analytical power of the QTOF MS platform to assess monoclonal antibodies quality attributes.

The biopharmaceutical industry is growing at a fast pace, making nowadays 20% of the pharma market. Within this market, therapeutic monoclonal antibodies (mAbs) are the dominant product class. With the patent expirations, biosimilars and, perhaps more relevant, biobetters, are in fast development. Thus, a comprehensive characterization at the molecular level of antibodies heterogeneity such as glycoforms, post-translational modifications (PTMs) and sequence variations is of utmost importance. Mass spectrometry (MS)-based approaches are undoubtedly the most powerful analytical strategies to monitor and define an array of critical quality attributes on mAbs. In this work, we demonstrate the analytical power of the Q-TOF MS platform for comprehensive and detailed analysis at molecular levels of an in-house produced mAb. This methodology involves minimal sample preparation procedures and provides an extensive collection of valuable data in a short period of time.

Exploring the regulatory levels of SUMOylation to increase crop productivity.

SUMOylation is an essential post-translational modification that affects several cellular processes, from gene replication to stress response. Studies using the SUMO (de)conjugation machinery have provided evidence regarding its potential to improve crop performance and productivity under normal and adverse conditions. However, the pleiotropic effect of SUMOylation can be a disadvantage in both situations, especially when considering unpredictable environmental conditions caused by climate changes. Here, we discuss the pleiotropic effects caused by disrupting the SUMOylation machinery, and new strategies that may help to overcome pleiotropy. We propose exploring the several regulatory levels of SUMOylation recently revealed, including transcriptional, post-transcriptional regulation by alternative splicing, and post-translational modifications. These new findings may provide valuable tools to increase crop productivity.

Ssu72 phosphatase is a conserved telomere replication terminator.

Telomeres, the protective ends of eukaryotic chromosomes, are replicated through concerted actions of conventional DNA polymerases and elongated by telomerase, but the regulation of this process is not fully understood. Telomere replication requires (Ctc1/Cdc13)-Stn1-Ten1, a telomeric ssDNA-binding complex homologous to RPA Here, we show that the evolutionarily conserved phosphatase Ssu72 is responsible for terminating the cycle of telomere replication in fission yeast. Ssu72 controls the recruitment of Stn1 to telomeres by regulating Stn1 phosphorylation at Ser74, a residue located within its conserved OB-fold domain. Consequently, ssu72∆ mutants are defective in telomere replication and exhibit long 3'-ssDNA overhangs, indicative of defective lagging-strand DNA synthesis. We also show that hSSU72 regulates telomerase activation in human cells by controlling recruitment of hSTN1 to telomeres. These results reveal a previously unknown yet conserved role for the phosphatase SSU72, whereby this enzyme controls telomere homeostasis by activating lagging-strand DNA synthesis, thus terminating the cycle of telomere replication.

Insights into the transcriptional and post-transcriptional regulation of the rice SUMOylation machinery and into the role of two rice SUMO proteases.

BACKGROUND: SUMOylation is an essential eukaryotic post-translation modification that, in plants, regulates numerous cellular processes, ranging from seed development to stress response. Using rice as a model crop plant, we searched for potential regulatory points that may influence the activity of the rice SUMOylation machinery genes. RESULTS: We analyzed the presence of putative cis-acting regulatory elements (CREs) within the promoter regions of the rice SUMOylation machinery genes and found CREs related to different cellular processes, including hormone signaling. We confirmed that the transcript levels of genes involved in target-SUMOylation, containing ABA- and GA-related CREs, are responsive to treatments with these hormones. Transcriptional analysis in Nipponbare (spp. japonica) and LC-93-4 (spp. indica), showed that the transcript levels of all studied genes are maintained in the two subspecies, under normal growth. OsSUMO3 is an exceptional case since it is expressed at low levels or is not detectable at all in LC-93-4 roots and shoots, respectively. We revealed post-transcriptional regulation by alternative splicing (AS) for all genes studied, except for SUMO coding genes, OsSIZ2, OsOTS3, and OsELS2. Some AS forms have the potential to alter protein domains and catalytic centers. We also performed the molecular and phenotypic characterization of T-DNA insertion lines of some of the genes under study. Knockouts of OsFUG1 and OsELS1 showed increased SUMOylation levels and non-overlapping phenotypes. The fug1 line showed a dwarf phenotype, and significant defects in fertility, seed weight, and panicle architecture, while the els1 line showed early flowering and decreased plant height. We suggest that OsELS1 is an ortholog of AtEsd4, which was also supported by our phylogenetic analysis. CONCLUSIONS: Overall, we provide a comprehensive analysis of the rice SUMOylation machinery and discuss possible effects of the regulation of these genes at the transcriptional and post-transcriptional level. We also contribute to the characterization of two rice SUMO proteases, OsELS1 and OsFUG1.

Synergistic Binding of bHLH Transcription Factors to the Promoter of the Maize NADP-ME Gene Used in C4 Photosynthesis Is Based on an Ancient Code Found in the Ancestral C3 State.

C4 photosynthesis has evolved repeatedly from the ancestral C3 state to generate a carbon concentrating mechanism that increases photosynthetic efficiency. This specialized form of photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the world's most productive crops. The C4 cycle is accomplished through cell-type-specific accumulation of enzymes but cis-elements and transcription factors controlling C4 photosynthesis remain largely unknown. Using the NADP-Malic Enzyme (NADP-ME) gene as a model we tested whether mechanisms impacting on transcription in C4 plants evolved from ancestral components found in C3 species. Two basic Helix-Loop-Helix (bHLH) transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C4NADP-ME promoter from maize. These proteins form heterodimers and ZmbHLH129 impairs trans-activation by ZmbHLH128. Electrophoretic mobility shift assays indicate that a pair of cis-elements separated by a seven base pair spacer synergistically bind either ZmbHLH128 or ZmbHLH129. This pair of cis-elements is found in both C3 and C4 Panicoid grass species of the PACMAD clade. Our analysis is consistent with this cis-element pair originating from a single motif present in the ancestral C3 state. We conclude that C4 photosynthesis has co-opted an ancient C3 regulatory code built on G-box recognition by bHLH to regulate the NADP-ME gene. More broadly, our findings also contribute to the understanding of gene regulatory networks controlling C4 photosynthesis.

The rice cold-responsive calcium-dependent protein kinase OsCPK17 is regulated by alternative splicing and post-translational modifications.

Plant calcium-dependent protein kinases (CDPKs) are key proteins implicated in calcium-mediated signaling pathways of a wide range of biological events in the organism. The action of each particular CDPK is strictly regulated by many mechanisms in order to ensure an accurate signal translation and the activation of the adequate response processes. In this work, we investigated the regulation of a CDPK involved in rice cold stress response, OsCPK17, to better understand its mode of action. We identified two new alternative splicing (AS) mRNA forms of OsCPK17 encoding truncated versions of the protein, missing the CDPK activation domain. We analyzed the expression patterns of all AS variants in rice tissues and examined their subcellular localization in onion epidermal cells. The results indicate that the AS of OsCPK17 putatively originates truncated forms of the protein with distinct functions, and different subcellular and tissue distributions. Additionally, we addressed the regulation of OsCPK17 by post-translational modifications in several in vitro experiments. Our analysis indicated that OsCPK17 activity depends on its structural rearrangement induced by calcium binding, and that the protein can be autophosphorylated. The identified phosphorylation sites mostly populate the OsCPK17 N-terminal domain. Exceptions are phosphosites T107 and S136 in the kinase domain and S558 in the C-terminal domain. These phosphosites seem conserved in CDPKs and may reflect a common regulatory mechanism for this protein family.

Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose-phosphate synthase and is required for a proper cold stress response.

Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here, we test the hypothesis that OsCPK17 plays a role in rice cold stress response by analysing OsCPK17 knockout, silencing and overexpressing rice lines under low temperature. Altered OsCPK17 gene expression compromises cold tolerance performance, without affecting the expression of key cold stress-inducible genes. A comparative phosphoproteomic approach led to the identification of six potential in vivo OsCPK17 targets, which are associated with sugar and nitrogen metabolism, and with osmotic regulation. To test direct interaction, in vitro kinase assays were performed, showing that the sucrose-phosphate synthase OsSPS4 and the aquaporin OsPIP2;1/OsPIP2;6 are phosphorylated by OsCPK17 in a calcium-dependent manner. Altogether, our data indicates that OsCPK17 is required for a proper cold stress response in rice, likely affecting the activity of membrane channels and sugar metabolism.

Selection of an Appropriate Protein Extraction Method to Study the Phosphoproteome of Maize Photosynthetic Tissue.

Often plant tissues are recalcitrant and, due to that, methods relying on protein precipitation, such as TCA/acetone precipitation and phenol extraction, are usually the methods of choice for protein extraction in plant proteomic studies. However, the addition of precipitation steps to protein extraction methods may negatively impact protein recovery, due to problems associated with protein re-solubilization. Moreover, we show that when working with non-recalcitrant plant tissues, such as young maize leaves, protein extraction methods with precipitation steps compromise the maintenance of some labile post-translational modifications (PTMs), such as phosphorylation. Therefore, a critical issue when studying PTMs in plant proteins is to ensure that the protein extraction method is the most appropriate, both at qualitative and quantitative levels. In this work, we compared five methods for protein extraction of the C4-photosynthesis related proteins, in the tip of fully expanded third-leaves. These included: TCA/Acetone Precipitation; Phenol Extraction; TCA/Acetone Precipitation followed by Phenol Extraction; direct extraction in Lysis Buffer (a urea-based buffer); and direct extraction in Lysis Buffer followed by Cleanup with a commercial kit. Protein extraction in Lysis Buffer performed better in comparison to the other methods. It gave one of the highest protein yields, good coverage of the extracted proteome and phosphoproteome, high reproducibility, and little protein degradation. This was also the easiest and fastest method, warranting minimal sample handling. We also show that this method is adequate for the successful extraction of key enzymes of the C4-photosynthetic metabolism, such as PEPC, PPDK, PEPCK, and NADP-ME. This was confirmed by MALDI-TOF/TOF MS analysis of excised spots of 2DE analyses of the extracted protein pools. Staining for phosphorylated proteins in 2DE revealed the presence of several phosphorylated isoforms of PEPC, PPDK, and PEPCK.

Screening for Abiotic Stress Tolerance in Rice: Salt, Cold, and Drought.

Rice (Oryza sativa) is the primary source of food for more than half of the world population. Most rice varieties are severely injured by abiotic stresses, with strong social and economic impact. Understanding rice responses to stress may help breeding for more tolerant varieties. However, papers dealing with stress experiments often describe very different experimental designs, thus making comparisons difficult. The use of identical setups is the only way to generate comparable data. This chapter is organized into three sections, describing the experimental conditions established at the Genomics of Plant Stress (GPlantS) unit of ITQB to assess the response of rice plants to three different abiotic stresses--high salinity, cold stress, and drought. All sections include a detailed description of the materials and methodology, as well as useful notes gathered from the GPlantS team's experience. We use rice seedlings as plants at this stage show high sensitivity to abiotic stresses. For the salt and cold stress assays we use hydroponic cultures, while for the drought assay plants are grown in soil and subjected to water withholding. All setups enable visual score determination and are suitable for sample collection along the imposition of stress. The proposed methodologies are simple and affordable to implement in most labs, allowing the discrimination of several rice genotypes at the molecular and phenotypic level.

Rice phytochrome-interacting factor protein OsPIF14 represses OsDREB1B gene expression through an extended N-box and interacts preferentially with the active form of phytochrome B.

DREB1/CBF genes, known as major regulators of plant stress responses, are rapidly and transiently induced by low temperatures. Using a yeast one-hybrid screening, we identified a putative Phytochrome-Interacting bHLH Factor (OsPIF14), as binding to the OsDREB1B promoter. bHLH proteins are able to bind to hexameric E-box (CANNTG) or N-box (CACG(A/C)G) motifs, depending on transcriptional activity. We have shown that OsPIF14 binds to the OsDREB1B promoter through two N-boxes and that the flanking regions of the hexameric core are essential for protein-DNA interaction and stability. We also showed that OsPIF14 down-regulates OsDREB1B gene expression in rice protoplasts, corroborating the OsPIF14 repressor activity observed in the transactivation assays using Arabidopsis protoplasts. In addition, we showed that OsPIF14 is indeed a phytochrome interacting factor, which preferentially binds to the active form (Pfr) of rice phytochrome B. This raises the possibility that OsPIF14 activity might be modulated by light. However, we did not observe any regulation of the OsDREB1B gene expression by light under control conditions. Moreover, OsPIF14 gene expression was shown to be modulated by different treatments, such as drought, salt, cold and ABA. Interestingly, OsPIF14 showed also a specific cold-induced alternative splicing. All together, these results suggest the possibility that OsPIF14 is involved in cross-talk between light and stress signaling through interaction with the OsDREB1B promoter. Although in the absence of stress, OsDREB1B gene expression was not regulated by light, given previous reports, it remains possible that OsPIF14 has a role in light modulation of stress responses.

Dps from Deinococcus radiodurans: oligomeric forms of Dps1 with distinct cellular functions and Dps2 involved in metal storage.

The DNA binding proteins from starved cells from Deinococcus radiodurans, Dps1-DR2263 and Dps2-DRB0092, have a common overall structure of hollow spherical dodecamers. Their involvement in the homeostasis of intracellular metal and DNA protection was addressed. Our results show that DrDps proteins are able to oxidize ferrous to ferric iron by oxygen or hydrogen peroxide. The iron stored inside the hollow sphere cavity is fully released. Furthermore, these proteins are able to store and release manganese, suggesting they can play a role in manganese homeostasis as well. The interaction of DrDps with DNA was also addressed. Even though DrDps1 binds both linear and coiled DNA, DrDps2 preferentially binds to coiled DNA, forming different protein-DNA complexes, as clearly shown by atomic force microscopy. DrDps1 (dimer and dodecamer) and DrDps2 can protect DNA against reactive oxygen species, although the protection occurs at different Fe to protein ratios. The difference between DrDps could be the result of the DrDps1 higher iron oxidation rate in the presence of hydrogen peroxide and its higher affinity to bind DNA than in DrDps2. Using cellular extracts obtained from D. radiodurans cultures, we showed that DrDps1 oligomers observed in in vitro conditions are also present in vivo. This indicates that DrDps1 has a structural dynamic plasticity that allows its oligomeric state to change between dimer, trimer and dodecamer. This in turn suggests the existence of a regulation mechanism that modulates the oligomer equilibrium and is dependent on growth stages and environmental conditions.

SUMOylation of the brain-predominant Ataxin-3 isoform modulates its interaction with p97.

BACKGROUND: Machado-Joseph Disease (MJD), a form of dominantly inherited ataxia belonging to the group of polyQ expansion neurodegenerative disorders, occurs when a threshold value for the number of glutamines in Ataxin-3 (Atx3) polyglutamine region is exceeded. As a result of its modular multidomain architecture, Atx3 is known to engage in multiple macromolecular interactions, which might be unbalanced when the polyQ tract is expanded, culminating in the aggregation and formation of intracellular inclusions, a unifying fingerprint of this group of neurodegenerative disorders. Since aggregation is specific to certain brain regions, localization-dependent posttranslational modifications that differentially affect Atx3 might also contribute for MJD. METHODS: We combined in vitro and cellular approaches to address SUMOylation in the brain-predominant Atx3 isoform and assessed the impact of this posttranslational modification on Atx3 self-assembly and interaction with its native partner, p97. RESULTS: We demonstrate that Atx3 is SUMOylated at K356 both in vitro and in cells, which contributes for decreased formation of amyloid fibrils and for increased affinity towards p97. CONCLUSIONS AND GENERAL SIGNIFICANCE: These findings highlight the role of SUMOylation as a regulator of Atx3 function, with implications on Atx3 protein interaction network and self-assembly, with potential impact for further understanding the molecular mechanisms underlying MJD pathogenesis.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing.

BACKGROUND: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. RESULTS: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. CONCLUSIONS: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.